The present invention relates to novel insulin analogues and their use in pharmaceutical compositions for the treatment of diabetes.
Insulin is a hormone which has a key role in the regulation of growth and metabolism in vertebrates. Severe metabolic derangements occur in the absence of insulin resulting from the failure of many cells to utilize glucose and amino acids normally. The inability to metabolize glucose leads in man to diabetes mellitus, a complex chronic metabolic disorder in which there is abnormal carbohydrate, fat and protein metabolism. In its most fully expressed clinical form, diabetes mellitus is characterized by an absolute or relative deficiency of insulin or insulin activity and is associated with glucosuria, ketonuria, growth arrest, and negative nitrogen balance. These conditions can ultimately lead to death from acute metabolic acidosis caused by unrestrained oxidation of fatty acids or inanition which results from the lack of sufficient lipid reserves needed to generate ketone bodies. Inanition is defined as a condition characterized by marked weakness, extreme weight loss, and a decrease in metabolism resulting from prolonged and severe insufficiency of food. Dorland's Illustrated Medical Dictionary, 25th Edition.
The discovery and purification of insulin in the 1920's and its association with diabetes mellitus provided the means to intervene in the disease. See, e.g., Bliss, The Discovery of Insulin (1983), University of Chicago Press, Chicago, Ill. Today, insulin administration to diabetic patients is the primary therapeutic means for controlling the disease.
Insulin is a ca 6000 dalton polypeptide which is composed of two short peptide chains, termed A and B, which are linked to each other by invariant disulfide bridges. In almost all insulins studied, the A chain, which is 21 amino acids long, also contains an internal disulfide bridge. The B chain is 30 amino acids in length. Like many eukaryotic proteins, insulin is synthesized in a precursor form which is post-synthetically processed to the mature two polypeptide chain active hormone.
The immediate precursor of insulin is proinsulin, a single chain polypeptide composed of the B and A chains linked to a connecting peptide of approximately 31 amino acids, termed the C-peptide, by adjacent pairs of basic residues. The order of the three peptides in the proinsulin molecule is NH.sub.2 -B chain-Arg-Arg-C-peptide-Lys-Arg-A chain-COOH. The translation product of insulin mRNA, however, is preproinsulin which is proinsulin that contains at its NH.sub.2 terminus a 24 amino acid largely hydrophobic signal peptide characteristic of proteins that are either transported through or inserted into cellular membranes.
Preproinsulin is synthesized in pancreatic beta cells located within the islets of Langerhans which are dispersed throughout the pancreas. Removal of the signal peptide occurs in the rough endoplasmic reticulum with the resulting fully folded oxidized proinsulin being transported to the Golgi apparatus for packaging into secretion granules. The folded proinsulin is stabilized by disulfide bonds. During maturation of the secretion granules, the folded proinsulin molecule is cleaved by specific proteases at the paired basic residues to liberate insulin and the C-peptide.
As discussed above, therapy for diabetes mellitus includes administration of controlled amounts of insulin to the diabetic patient. The insulin so administrated has, for the most part, been obtained from animal pancreases, notably bovine and porcine. Bovine and porcine insulins function to maintain hormonal homeostasis in the same way as human insulin with about the same potency but, because they are foreign proteins, can elicit an immunological response which diminishes their usefulness. More recently, human insulin, generated by recombinant DNA techniques, has been added to the therapeutic armamentarium. The use of human insulin, produced by recombinant DNA or other techniques, is not likely to produce the adverse immunological problems attendant the use of animal insulins. Even with the availability of natural human insulin, however, administration of the hormone to diabetics has not been always sufficient to restore normal metabolism. There is thus a need for alternative insulins with better activity or other means of therapy for diabetes.
U.S. application Ser. No. 074,558 discloses a superactive human insulin analogue, [10-Aspartic Acid-B] human insulin, which has increased activity over natural human insulin. Specifically, [10-Aspartic Acid-B] human insulin was determined to be 4 to 5 times more potent than natural insulins. U.S. application Ser. No. 273,957 and International Application Serial No. PCT/US88/02289 disclose other superactive insulin analogues, des-pentapeptide (B26-B30)-[Asp.sup.B10, Tyr.sup.B25 -.alpha.-carboxamide] human insulin, (B26-B30)-[Glu.sup.B10, Tyr.sup.B25 -.alpha.-carboxamide] human insulin, and further insulin analogs of the formula des(B26-B30)-[X.sup.B10, Tyr.sup.B25 -.alpha.-carboxamide] human insulin, in which X is a residue substituted at position 10 of the B chain. These insulin analogues have potencies anywhere from 11 to 20 times that of natural human insulin. All of the above-described insulin analogues involve amino acid substitutions along the A or B chains of natural human insulin, which increase the potency of the compound or change other properties of the compound.
None of the current insulin delivery routes of natural insulin and known insulin analogues exactly mimic insulin secretion from the normal pancreas. Normally, insulin enters the splanchnic venous circulation, thereby exposing the liver to higher concentrations of insulin than those to which peripheral tissue is exposed. With standard subcutaneous administration of insulin, plasma glucose concentrations may be normalized, but glucose recycling and the production and utilization of protein and lipids may not be. In addition, peripheral vascular tissues are exposed to higher than normal insulin concentrations. Although the long-term effects of these metabolic abnormalities remains to be defined, there is considerable evidence that peripheral hyperinsulinemia may be a significant risk factor for the development of atherosclerosis.
Hepatospecific insulin analogues, or those which are more active in the liver than in adipose tissue, offer several advantages over currently available insulin therapy. Using such analogues it may be possible to obtain preferential hepatic uptake during peripheral subcutaneous administration, thereby mimicking more closely the metabolic balance between the liver and peripheral tissues. Although attempts to mimic this pattern with intraperitoneal injection of insulin have been undertaken, this technique has the potential disadvantages of difficultly for intraperitoneal access and risk of peritonitis. Hepatospecific insulin achieves the same effect as intraperitoneal insulin, without these increased risks.